The purpose of this study is to define the Male, naive, HIV-infected volunteers were assigned either to tenofovir-DF/emtricitabine/efavirenz (Group_T) or abacavir/lamivudine/efavirenz (Group_A). Platelet Activating Factor (PAF) levels and metabolic enzymes together with HIV-implicated cytokines  and VEGF were determined for a 12-month period. Differences within each group were determined by non-parametric Friedman and Wilcoxon test, while the differences between the groups were checked by ANOVA repeated measures.rd month as well as elevated Lp-PLA2 activity for abacavir-containing regimen.Both ART regimens present pronounced effect on inflammatory mediators, resulting in decreased PAF levels and Lipoprotein-associated phospholipase A2 (Lp-PLA2) activity for tenofovir-containing regimen and same as baseline PAF levels with a peak though at the 3Studies regarding the effect of first-line ART regimens on inflammation may be beneficial in preventing chronic morbidities during HIV-treatment. From this point of view, the present study suggests an anti-inflammatory effect of tenofovir-containing ART, while the temporary increase of PAF levels in abacavir-containing ART may be the link between the reported cardiovascular risk and abacavir administration. The progress of ART over the years has turned HIV-infection from lethal into a chronic disease. However, immune activation and inflammation are lying behind effectively treated patients causing various non-AIDS morbidities as cardiovascular or renal diseases and cancers . ConsequDuring HIV-infection specific cells of the immune system become targets of the virus and secrete in response several inflammatory factors as cytokines and chemokines . The effde novo, responsible for the constant PAF biosynthesis, and remodeling, activated as a direct response to acute inflammatory processes [Platelet Activating Factor (PAF) is one of the most potent inflammatory factors and a significant signaling molecule of the immune system, considered as a primitive and universal cellular mediator ,12 that rocesses , with kerocesses ,17, resprocesses .in vitro inhibitory effect against PAF activity [PAF is thought to be implicated in the progression of HIV-infection as studies have revealed that HIV-infected monocytes overexpress PAF through Tat protein ,20. It wactivity  while maactivity -28. Patiactivity . It has activity . The impactivity ,32 as weactivity .in vivo effect of two first line ART regimens on PAF levels in HIV naive patients along with several already established inflammatory biomarkers implicated in HIV-infection. The hypothesis states that tenofovir-DF/emtricitabine/efavirenz would down regulate PAF levels in contrast to abacavir/lamivudine/efavirenz based on preliminary data concerning the effect of the above regimens on PAF enzymes [The scope of this paper is to determine for the first time the  enzymes ,35.rd Internal Medicine Department-Infectious Diseases Unit, Red Cross General Hospital, Athens, Greece. All participants were male, treatment naïve, and asymptomatic HIV-infected patients as determined by the presence of antibodies against HIV measured by enzyme-linked immunosorbent assay (ELISA) and confirmed by Western blot. All patients were at CDC A2 clinical stage and fulfilled the criteria for ART initiation according to the European [Study enrollment begun after obtaining approval from the scientific board of the Red Cross General Hospital of Athens in Greece and all volunteer patients have signed the informed consent according to the Declaration of Helsinki. The volunteers (n = 18) were recruited from the 3European  and InteEuropean . The patThe isolation and purification of PAF was according to the method of Demopoulos et al. . BrieflyThe isolations of plasma, platelets (HPs) and leukocytes (HLs) were carried out as previously described . BrieflyDithiothreitol-insensitive PAF-CPT and lyso-PAF-AT activity assays were performed on the homogenates of leukocytes and platelets. PAF-AH activity assays were performed on the homogenates of leukocytes and platelets as well as Lp-PLA2 activity assays on plasma as previously described . The enzClinical biochemical markers were measured by a Siemens Dimension RxL automatic analyzer. CD4 cell counts were defined using Tetra One System on the EPICS XL flow cytometer, while viral load was determined using the Versant HIV-1 RNA 3.0 assay. Plasma levels of IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNFα and VEGF were determined on a BD FACS Canto II Flow cytometer using CBA flex set cytokine kit and data analysis was performed by FCAP Array DIVA software. Cytokines are expressed as pg/mL of plasma.th-75th). Differences within each group during the overall 12-month treatment were determined by non-parametric Friedman (displayed as ptime) analysis. Wilcoxon test was used to compare the difference of specific time point with the baseline values within each group (displayed as pspecific-time-point). After ranking the values, the differences between the groups were checked by repeated measures ANOVA and are displayed as Pintervention (Pint) for the overall and Pspecific-time-point for each time point in particular. Viral load changes are reported in a logarithmic scale for convenience and not in order to normalize the value. Statistical significance was considered as p < 0.05.Normal distribution was checked by P-P graphs using Shapiro-Wilk criterion. The results are expressed as median values and interquartile range (25int.s > 0.05).Anthropometric and biochemical characteristics of patients are shown in Additional file time_A/T < 0.001), while CD4 cell counts are gradually increased (ptime_A/T < 0.001) even from the 1st month of treatment. White blood cell count remains stable in both groups , belonging to the minimum level of the normal range.In both groups, viral load is progressively reduced during the study period , LDL  and HDL  are increased while triglycerides remain stable (ptime_T = 0.170) in Group_T and are increased in Group_A (ptime_A = 0.001).Despite the variations in the lipid profile of each group, the values remain within the normal range. Particularly, in both groups total cholesterol (ptime_T = 0.010) and especially they are decreased at the 1st, 3rd, 6th and 12th month . Bound PAF levels in Group_A are also differentiated during the study period (ptime_A = 0.028), with a single increase at the 3rd month (p3 = 0.016), (Table int = 0.357), however a differentiation is achieved at the 3rd month (p3_A-T = 0.038) and a borderline one at the 12th month (p12_A-T = 0.083);  while in Group_A they remain stable throughout the treatment period (ptime_A = 0.719), with no differentiation at any time point for both groups nor between them (Pint = 0.917),  with gradually reductions from the 1st to the 12th month . In contrast, in Group_A it is borderline differentiated through the study period (ptime_A = 0.085), achieving however an increase, at the 12th month (p12 = 0.006). Although the comparison of the two groups presents no overall differentiation (Pint = 0.304), there is a significant one at the 12th month (p12_A-T = 0.016). Regarding the specific activity of lyso-PAF-AT, in Group_T it is differentiated during the 12-month treatment (ptime_T = 0.002) with significant decreases from the 6th to the 12th month . On the contrary, in Group_A, no difference is observed throughout the study period (ptime_A = 0.141), however, an increase is detected at the 3rd month (p3 = 0.037);  with a reduction, however, at the 12th month (p12 = 0.047). Besides, the specific activity of lyso-PAF-AT in Group_T remains stable through the 12-month period (ptime_T = 0.416), decreasing though at the 9th month (p9 = 0.039), while in Group_A it is differentiated during the study period (ptime_A = 0.041) with a significant peak at the 3rd month of treatment (p3 = 0.010); ; , while in Group_A, it is increased at the 9th month (p9 = 0.002), , with significant decreases from the 6th to the 12th month . In contrast, in Group_A, the specific activity of PAF-AH is not differentiated during the study period, achieving however a significant peak at the 3rd month (p3 = 0.049), , with significant reductions from the 6th to the 12th month , , although a single increase of TNFα levels in Group_A (p1 = 0.027) is observed at the 1st month of treatment, .Our team has already shown that tenofovir-DF/emtricitabine/efavirenz has great inst PAF  and decrinst PAF  while aby effect  against rocesses . The prerd month. This differentiation is probably attributed to the peak of lyso-PAF-AT activity at the 3rd month in Group_A in parallel with the continual decrease of PAF biosynthesis in Group_T. There is also a trend of difference at PAF levels noted at the 12th month, which may be credited to the significant difference at the 12th month of PAF-CPT as well to the opposite effect of the two regimens on both biosynthetic enzymes. The above results of higher PAF levels and metabolic enzymes’ activity, seen at certain time points, support a possible direct effect of abacavir than an indirect effect of immune reconstitution as it occurs from the white blood cell count.In the present study, PAF levels in HIV patients are measured for the first time, resulting surprisingly to lower values than the average population although the activity of PAF biosynthetic enzymes is much higher . One mayth and the 12th month. Moreover, in Group_T the decrease of Lp-PLA2 at the 9th and the 12th month comes in accordance with the decrease in PAF levels during the 12-month period. In Group_A the increase of PAF levels at the 3rd month may be the reason of Lp-PLA2 raise at the 9th month or this raise possibly reflects a direct effect of abacavir/lamivudine/efavirenz on this enzyme.In this study the activity of Lp-PLA2, a well-known cardiovascular risk marker and sensitive to PAF levels , is highst month in Group_A. The elevated TNFα levels may assist to the increased PAF levels at the 3rd month, knowing that TNFα induces PAF biosynthesis and vice versa[The inflammatory cytokines IL-1β, IL-6, IL-10, IL12p70 and TNFα are implicated both at the pathogenesis and the morbidities of HIV infection. In the present study, cytokines’ levels are higher than healthy population indicating the immune state of these HIV patients . Cytokinice versa,46.In conclusion the present study points out the effect of ART regimens to the persistent inflammatory state observed in the setting of suppressive antiretroviral treatment. The tested regimens caused an opposite outcome on PAF levels and on Lp-PLA2 activity. Since PAF is a potent inflammatory factor, associated with pro-atherosclerotic complications, the above results indicate a potent anti-inflammatory role for tenofovir based regimen while suggest PAF as the missing link between the abacavir based regiment and the reported cardiovascular risk.The main limitation of this research study is the number of the samples. The reason is the chosen methods for PAF which are quite strenuous and time consuming but also more sensitive and trustful than other rapid methods. Female patients have not been used in the study as the menstrual cycle affects the metabolism of PAF.The authors declare no competing Interests. The research has been funded from the Hellenic Society for the Research, Study and Education in Infectious Diseases.V.D.P is responsible for the authorship, the experiments and the data analysis. G.S and A.B.T participated at the experiments and the data analysis. M.C, N.M and N.T designed the selection of patient samples, provided the clinical data for analysis and contributed to the interpretation of data, revising drafts critically for important intellectual content and final approval of the version to be published. E.F conducted the data analysis and reviewed the manuscript. S.A critically reviewed the manuscript. C.A.D and M.C.L conceived and supervised the whole study. All authors read and approved the final manuscript.Anthropometric and biochemical characteristics of ART groups.Click here for fileFree PAF levels and metabolic enzymes in platelets of ART groups.Click here for fileCytokines and VEGF levels in HIV-infected patients.Click here for file


L-carnitine (LC) plays an important physiologic role in lipid metabolism. To date, no clinical study has been performed to examine the effect of LC supplementation on the lipid status of coronary artery disease (CAD) patients. The aim of this study was to investigate the lipid lowering effects of LC supplementation (1000 mg/d) in CAD patients.n = 24) and to the LC (n = 23) groups. The intervention was administered for 12 weeks. The levels of LC, lipid profiles, and antioxidant enzyme activity  were measured.CAD patients were identified by cardiac catheterization as having at least 50 % stenosis of one major coronary artery. Forty-seven subjects were recruited and randomly assigned to the placebo (P < 0.01), high density lipoprotein-cholesterol , and apolipoprotein-A1  than those in the placebo group at week 12. Triglyceride (TG) level was slightly significantly reduced  and the level of LC was negatively correlated with TG and apolipoprotein-B (Apo-B), and positively correlated with HDL-C and Apo-A1 after LC supplementation. Additionally, SOD activity was significantly negatively correlated with lipid profiles  after supplementation.The subjects in the LC group had significantly higher SOD activity (20.7 ± 4.2 versus 13.1 ± 2.9 U/mg of protein, LC supplementation at a dose of 1000 mg/d showed significantly increased in HDL-C and Apo-A1 levels and a slight decrease in TG levels but no other changes in other lipids in CAD patients, and this lipid-lowering effect may be related to its antioxidant ability. Further studies should be conducted to define an optimal dose of LC for lipid-lowering in patients with CAD.NCT01819701Clinical Trials.gov Identifier:  L-carnitine (LC) plays an important physiologic role in lipid metabolism. LC may carry long-chain fatty acids across the inner mitochondrial membrane for β-oxidation and adenosine triphosphate (ATP) production , 2. ReceA number of clinical studies have been conducted to examine the lipid-lowering effects of LC supplementation in patients with renal disease , 12–17, NCT01819701). This clinical trial started recruiting subjects at January 2013, and the last subject was completed in February 2014. Each subject provided written informed consent to participate in the study. A total of 47 CAD patients were recruited to this study and randomly assigned to the placebo (n = 24) or to the LC  group. The supplements, placebo (starch) and LC capsules were provided by New Health Taiwan Co., Ltd . At the beginning of the study, the investigators instructed the subjects to take two capsules daily (LC supplements 500 mg b.i.d). To monitor the subject compliance, we asked the subjects to return the supplied bag of capsules every 4 weeks to verify the capsule count. The intervention was administered for 12 weeks. The sampling and trial profiles along with the number of subjects who completed the study in each group are presented in Fig. A single-blind, randomized, parallel, placebo-controlled trial was conducted. CAD patients were diagnosed by cardiac catheterization (at least 50 % stenosis of one major coronary artery) or by percutaneous transluminal coronary angioplasty (PTCA) for stable condition. We excluded the patients with diabetes, liver, or renal diseases, along with those undergoing acenocoumarol, thyroid hormone, warfarin, and a high dose of statin therapy  or currently using vitamin supplements. The study was approved by the Institutional Review Board of Taichung Veterans General Hospital, Taiwan and the clinical trial was registered at Clinical Trials.gov NCT0819701. T2). General obesity was defined as body mass index ≥ 27 kg/m2 [At baseline, we measured the subjects’ blood pressure, body weight, height, and waist circumference, and then calculated the body mass index . The samples were centrifuged at 3,000 rpm for 15 minutes at 4 °C, and the serum was separated. Serum total cholesterol (TC), triacylglycerol (TG), low density lipoprotein-cholesterol (LDL-C), and high density lipoprotein-cholesterol (HDL-C) were measured using an automated biochemical analyzer . The levels of apolipoprotein A-1 (Apo-A1) and apolipoprotein-B (Apo-B) were measured using the polyethylene glycerol (PEG) enhanced immunoturbidimetric assay method . The level of LC was measured by the enzyme-linked immunosorbent assay (ELISA) method using commercially available kits  according to the instructions made available by the suppliers.The antioxidant enzyme activity  were measured in the fresh red blood cells (RBCs) samples. The RBCs were washed with normal saline after removing the plasma, and then diluted with 25x sodium phosphate buffer for measurement. The methods for measuring SOD activity have been described previously . The prot-test or Wilcoxon signed rank test was used to compare the data before and after supplementation within the group. For categorical response variables, differences between the two groups were assessed by the Chi-square test or Fisher's exact test. Simple linear regressions were used to examine the correlations between the levels of LC (as a dependent variable) and lipid profiles after supplementation. Pearson correlations were used to examine the correlations between the antioxidant enzyme activity and lipid profiles after LC supplementation and the correlations between the changes  in antioxidant enzyme activity and lipid profiles. Statistical significance was set at P < 0.05. All statistical analyses were performed using SigmaPlot software .Means and standard deviations (s.d.) were calculated for all data. Kolmogorov-Smirnov test was used to examine the normal distribution of variables. Student’s t-test or the Mann-Whitney rank sum test was used to compare mean values for continuous variables between the placebo and LC groups. Paired n = 19; LC, n = 20). The baseline characteristics of the subjects are shown in Table A total of 39 CAD subjects completed the 12-week interventional study  and SOD activity  compared with those in the placebo group at week 12 as well as after LC supplementation . The lipid profiles after supplementation are shown in Fig. P = 0.03) and Apo-A1  than those in the placebo group at week 12. After LC supplementation, the level of TG was slightly reduced but did not achieve statistical significance . However, no significant change in the levels of TC, LDL-C, and Apo-B after 12 weeks of LC supplementation. Notably, the level of Apo-A1 was decreased in the placebo group after 12 weeks intervention ; however, the result was not statistically significant.The subjects in the LC group had significantly increased LC level . Although not statistically significant, LC level correlated with Apo-B , HDL-C  and Apo-A1  after 12 weeks of supplementation.The correlations between the levels of lipid profiles and LC after supplementation and these results are shown in Fig. r = -0.34, P = 0.03), TG , and Apo-B  after 12 weeks of LC supplementation. Furthermore, we assessed the correlations between the changes  in antioxidant enzyme activity and lipid profiles , LDL-C , and Apo-B .The correlations between the antioxidant enzyme activity and lipid profiles after LC supplementation are shown in Table β = 0.72, P < 0.01) and reduced oxidative stress and inflammatory status in the patients with CAD after supplementation [Significant lipid-lowering effects of LC supplementation have been demonstrated in hemodialysis patients , 17. Naientation , 27. In Apolipoproteins, such as Apo-B and Apo A-1, are the major apolipoproteins associated with LDL-C and HDL-C, respectively . In thisn = 13; LC, n = 8) with hyperlipidemia  [In the present study, TG level decreased after supplementation, but the reduction did not achieve statistical significance, which may have been attributed to the stable CAD subjects and the limited number of subjects  . Due to  mmol/L) , 33, we  mmol/L) , 27. The mmol/L) .The strength of this study is that it is the first clinical study to investigate the lipid-lowering effects of LC supplementation in CAD patients, and we measured the levels of LC, lipid profiles, and antioxidant enzyme activity. This study provides direct evidence to clarify the relationship among the levels of LC, lipid, and antioxidant ability; however, longer intervention studies with larger sample sizes should be performed to confirm the lipid-lowering effects after LC intervention in patients with hyperlipidemia. There are some limitations of the present study that should be mentioned. First, the number of participants was small; however, we performed the post hos calculations to examine the statistical power for lipid profiles. The statistical power of the differences of TG, HDL-C, and Apo-A1 were 0.97, 0.86, and 0.86, respectively. Second, a few of the subjects had hyperlipidemia in the LC group; thus, the detection of the lipid-lowering effects in these subjects may have been limited. Moreover, an optimal dose of LC for lipid-lowering should be defined. Further studies should also investigate the effect of LC on NADPH oxidase activity, HMG-CoA reductase activity and TG synthesis to clarify if LC lowers lipids by enhancing mitochondrial fatty acid oxidation or by reducing oxidative stress.In conclusion, LC supplementation at a dose of 1000 mg/d exhibited significant increases in HDL-C and Apo-A1 levels and a slight decrease in TG levels, but no other changes in other lipids were noted in CAD patients. This lipid-lowering effect may be related to its antioxidant ability.


Caenorhabditis elegans. Multiple toxicity end points including their mortality, behaviour, reproduction, in vitro distribution and expression of stress response mtl-1 and sod-1 genes were observed to evaluate safe application of ZnO-NPs. C. elegans were exposed to 10, 50, and 100 nm ZnO-NPs (0.1 to 2.0 g/l). Application of 10 nm ≥0.7g/l adversely affects the survivability of worms and was significantly not affected with exposure of 50 and 100 nm ≤1.0 g/l. However, reproduction was affected at much low concentration as compared to their survivability. LC50 was recorded 1.0 ± 0.06 (g/l) for 100 nm, 0.90 ± 0.60 for 50 nm and 0.620 ± 0.08 for 10 nm. Expression of mtl-1 and sod-1 was significantly increased with application of 10 nm ≥0.7g/l and significantly unaffected with exposure of 50 and 100 nm at the same concentration. ZnO-NPs (10 nm) had shown even distribution extended nearly the entire length of the body. The distribution pattern of ZnO-NPs indicates that the intestine is the major target tissues for NP toxicity. Study demonstrates that small-sized (10 nm) ZnO-NPs ≥0.7g/l is more toxic than larger-sized particles. This may be suggested on the basis of available data; application of 50 and 100 nm ≤1.0 g/l ZnO-NPs may be used to the environment as this shows no significant toxicity. However, further calibration is warranted to explore safe dose on soil compartments prior to their field application.Zinc oxide nanoparticles (ZnO-NPs) are increasingly receiving attention due to their widespread application in cosmetics, pigments and coatings. This has raised concerns in the public and scientific communities regarding their unexpected health effects. Toxicity effect of ZnO-NPs on the environment was assessed in the present study using  Porcellio scaber, Caenorhabditis elegans and earthworms Eisenia fetida and Lumbricus rubellus, respectively. Furthermore, researches are needed to provide insight into the toxicological effect of exposure to NPs on organisms especially living in the soil. Auffan et al. [2, Ag and CeO2; toxicity is at least partly due to the specific properties related to the small size and consequent high surface activity of NPs, while their effects may be further enhanced by the release of the free metal ions. If the free ions are more toxic than the original particles, this process of dissolution is likely to lead an increase of the overall toxicity. Speciation of metal NPs is soils is not yet clearly understood. Some works talk about the distribution of Zn, from ZnO-NPs in soil is the most liable fractions (those accessible to inhabiting soil organism) and differences between ZnO-NPs and derivate ion Zn2+ [Daphnia magna, freshwater microalgae, mice and even human cell [Nanotechnology is rapidly expanding the field of science with continuous development of nanomaterials and their industrial application. Nanoforms of metals, metal oxides, carbon-based materials and biopolymers are being used in several industries including diagnosis, drug delivery, cosmetics, sunscreens, food, paints, electronics, sports, imaging, etc. Among them, zinc oxide nanoparticles (ZnO-NPs) are most exploited at nano-dimension level. They are abundantly used nanomaterials in cosmetics and sunscreens as they exhibit high catalytic efficiency, as well as strong absorption ability for UV light. This makes them transparent and more aesthetically acceptable compared to their bulk counterpart as suggested by Schilling et al. . ZnO-NPsn et al.  reportedion Zn2+ . Howeverman cell –17. Thesman cell  observedman cell  also obsman cell . Jeng anman cell  also obsC. elegans using multiple toxicity end points to record optimal dose and size of these NPs for their safe application.The increased production and use of ZnO-NPs enhances the probability of their exposure in occupational and environmental settings. Although this NP has great commercial importance and present in various commercial products, so therefore is growing concern in public and scientific communities regarding their unanticipated and adverse health effects. A significant portion of ZnO-NPs may be expected to be released in sewage sludge via waste water. Varying portions of sewage sludge were disposed in landfills and incinerated or applied to agricultural land . TherefoZnO-NPs  were purchased from Sigma Aldrich Chemical . Particles were labelled as suggested by Tachikawa et al.  with fluC. elegans Bristol strain N2 was obtained from Caenorhabditis Genetic Centre (CGC), USA, and culture was maintained on nematode growth medium (NGM) plates seeded with Escherichia coli strain OP50 at 20 °C, using the standard method [The wild-type d method . Young ad method . Nematodd method  and buffToxicity end points including LC50 (probit method), mortality and mobility were observed after 24-h incubation with exposure of NPs, and juveniles were recorded at 48 h. Visually dead worms were sorted out with a platinum wire under stereo microscope.mtl-1  and sod-1(anti oxidant enzyme) gene expression. Standard procedures were followed as suggested by Roh et al. [After 24-h incubation with exposure of NPs, nematodes were harvested for the analysis of After exposure of ZnO-NPs, fluorescence distribution images were observed by using fluorescence microscope equipped with a peltier cooled charge-coupled camera. Both differential interference contrast (DIC) and epi-fluorescence images were taken. Filter set with maxima of 460 nm was used for visualization of fluorescence.p values used to decide statistical significance based on the somewhat arbitrary choice of level were often set at 0.05 except for mortality and behaviour (0.01).Results are the means of three replicates. Two-way analysis of variance (ANOVA) was performed by using the SPSS 10.5 software. The objective of statistical analysis was to determine any significant differences among the parameters analysed in different treatments to record optimal dose and size of ZnO NPs for their safe application. C. elegans to ZnO-NPs caused an increase in percentage of mortality with an increase in concentration (0.1 to 2.0g/l) and decrease in size of NPs , helps in metal detoxification and homeostasis. It is ubiquitous in most of the animals. A cell-specific factor, as well as developmentally modulated and metal response pathways controls their transcription. Due to their inducibility to transition metals, they are considered as an important specific biomarker to detect organism response to heavy metals. They have been involved in the defence against reactive oxygen species and also in the homeostatic regulation of heavy metals. Otuska [mtl-1 gene may serve as significant model for investigating the toxicity of ZnO-NPs and mechanism to cellular response of uptake of heavy metals.Gene expression may consider as high sensitivity and mechanistic values to diagnose environmental contamination. Nowadays, environmental stress response in toxicology was increasingly recorded by using gene profiling by many workers including Snell et al. , Lee andanalysis . Low-mol. Otuska  assumed sod-1 gene, which is abundantly in cells throughout the body. This enzyme attaches the molecules of zinc to break down toxic-charged oxygen molecules called superoxide radicals. There may be the possibility of high accumulation of harmful superoxide radicals with exposure of small-sized NPs (10 nm) in the present study. This may responsible for production of other types of toxic radicals including increased cell death or formation of aggregates of misfolded super oxide dismutase. No significant change was observed in expression of mtl-1gene for all ZnO-NPs in the present study. However, low expressions were recorded for 100 nm in all treatments (Fig. C. elegans for smaller-sized particles. Relative band density of PCR products for mtl-1 enhanced for 10 nm at exposure of ≥0.5g/l and above that was most pronounced and significantly increased by 1.5 to 2.5. On the other hand, 50 and 100 nm band density almost remains constant. It appears that the exposure of 10 nm ZnO-NPs ≥0.4g/l increases the expression of mtl-1 gene and that is responsible for uptake of Zn+2. Even, the expression of mtl-1 gene was significantly increased in all treatments of 10 nm ZnO-NPs. Similarly, generation of reactive oxygen species was also significantly increased with an increase in concentration of 10 nm ZnO-NPs (Fig. mtl-1 and sod-1 may be involved in the metabolism of ZnO-NPs and/or toxicity in C. elegans. Expression of both genes may consider as an early warning signal, relating a molecular level response to determine environmental stress of ZnO-NPs and their ecological effects. This also represents a substantial challenge to correlate validated toxicity end points including induced gene expression. In general, ZnO-NP toxicity may results from two more replacement of essential metals at protein active sites in 10 nm ZnO-NPs and may be due to the generation of reactive oxygen species at large for smaller-sized particles that damage macro-molecules, including DNA and proteins and thus may impair cellular function.Similarly, the making of superoxide dismutase is instructed by nts Fig. . This maNPs Fig. . Data suC. elegans after exposure to ZnO-NPs showed high pixel intensities with increase in concentration and decrease in size of NPs. The highest pixel intensity was recorded with exposure of 10 nm NPs and shows more even distribution of fluorescence. However, at exposure of 100 nm (Fig. C. elegans, fluorescence extended nearly the entire length of the body(Fig. C. elegans confirmed the multiple biological functions in a wide range of cells and tissues by small nanoparticles, whereas an elevated intensity localized in particular area at exposure of 50 and 100 nm ZnO-NPs suggest the strategy that they accumulate NPs as “reservoir”. It stores extra Zn and releases it to support the organism’s need when Zn deficiency situation occurs as reported by Hongbo [C. elegans was almost similar. However, subtle difference was recorded at higher dose. The distribution pattern of ZnO-NPs may indicate that intestine is a major target tissue for NP toxicity. The fluorescence was also observed in eggs; a large number of eggs were recorded in 10-nm exposed worms (Fig. C. elegans and observed fluorescence effects may be due to high rate of biotransformation of small-sized 10 nm NPs. The findings of the study may be helpful to determine the safe application of ZnO-NPs in the environment.Remarkable increase in fluorescence was observed in all treatments compared to controls. Fluorescence distribution images of  nm Fig.  and 50 n nm Fig.  ZnO-NPs,bodyFig. . This may Hongbo . The disrms Fig. a–c. It iC. elegans. We conclude that the activity of worms was not adversely affected with exposure of 50 and 100 nm ZnO-NPs ≤1.0 g/l, while small-sized particles 10 nm ≥0.7g/l affects them at large as shown by an increase in the expression of mtl-1 and sod-1 gene. Distributional pattern of ZnO-NPs reveals that the intestine is the major target tissues for NP toxicity. The application of 50 and 100 nm ZnO-NPs may be safe to the environment in comparison to 10 nm. However, further calibration and validation of identified dose are warranted to explore more dose-response toxicity assessment and their kinetics of in vivo distribution.The present study was focussed to optimize the safe application of ZnO-NPs by assessing multiple toxicity points and in vitro distribution in nanoparticle-exposed


The response and susceptibility to astroglial degenerations are relevant to the distinctive properties of astrocytes in a hemodynamic-independent manner following status epilepticus (SE). Since impaired mitochondrial fission plays an important role in mitosis, apoptosis and programmed necrosis, we investigated whether the unique pattern of mitochondrial dynamics is involved in the characteristics of astroglial death induced by SE. In the present study, SE induced astroglial apoptosis in the molecular layer of the dentate gyrus, accompanied by decreased mitochondrial length. In contrast, clasmatodendritic (autophagic) astrocytes in the CA1 region showed mitochondrial elongation induced by SE. Mdivi-1  effectively attenuated astroglial apoptosis, but WY14643  aggravated it. In addition, Mdivi-1 accelerated clasmatodendritic changes in astrocytes. These regional specific mitochondrial dynamics in astrocytes were closely correlated with dynamin-related protein 1  phosphorylation, not optic atrophy 1  expression. To the best of our knowledge, the present data demonstrate for the first time the novel role of DRP1-mediated mitochondrial fission in astroglial loss. Thus, the present findings suggest that the differential astroglial mitochondrial dynamics may participate in the distinct characteristics of astroglial death induced by SE. Mitochondria are morphologically dynamic organelles that are essential for maintaining cell function, growth, and survival . All reagents were obtained from Sigma-Aldrich , except as noted.Male Sprague-Dawley rats (7 weeks old) were obtained from Daehan Biolink (South Korea). The animals were provided with a commercial diet and water 2 and placed in a stereotaxic frame. A brain infusion kit 1  was implanted into the right lateral ventricle , and connected to an osmotic pump  containing: (1) vehicle; (2) Mdivi-1 (50 μM); or (3) WY 14643 (150 μM). Mdivi-1 or WY14643 pretreatment did not affect the seizure susceptibility or its vulnerability in response to pilocarpine and animal survival rates following SE . To reduce peripheral effects of pilocarpine, Atropine methylbromide  was injected 20 min before a single dose of pilocarpine. Animals were maintained in SE for 2 h, after which diazepam  was administered to terminate seizure activity, and repeated, as needed. After SE, all animals were observed in the small animal intensive care units  and given 5% dextrose in lactate Ringer solution . To prevent drying of eyes, an ocular lubricant was applied. Animals were continuously monitored and injected with 5% dextrose in lactate Ringer solution at 4 h interval when needed. Next day, animals were fed moistened high-fat rodent chow and apple slices on the floor cage. As controls, age-matched normal rats were treated with saline instead of pilocarpine.2/area) were selected from the stratum radiatum of the CA1 field and the molecular layer of the dentate gyrus. Each image was normalized by adjusting the black and white range of the image using AxioVision Rel. 4.8 Software. Fluorescent intensity was then standardized by setting the threshold levels (mean background intensity obtained from 5 image input). Fluorescent intensity values were corrected by subtracting the average values of background noise (threshold level). Manipulation of the images was restricted to threshold and brightness adjustments to the whole image.Under urethane anesthesia , rats were transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer . After postfixation in the same fixative for 4 h, brains were infiltrated with 30% sucrose and sectioned with a cryostat at 30 μm. Sections were incubated overnight at room temperature in a mixture of primary antisera (Table 5 μm2) were selected from the stratum radiatum of the CA1 field and the molecular layer of the dentate. Cells were counted on 20× images using AxioVision Rel. 4.8 Software. Results are presented as means ± S.D. Individual mitochondrion length in astrocytes (n = 20/section) was also measured by using AxioVision Rel. 4.8 Software or ZEN lite  software following 3D-reconstruction . After regions were outlined, areas of interest . First, we confirmed the regional specific vulnerability of astrocytes to SE insults. Consistent with our previous studies  and WY14643 , but WY14643 reduced it (0.62 μm) in astrocytes of non-SE animals, as compared to vehicle . In contrast, WY14643 infusion increased the number of Ki-67 positive astrocytes in the CA1 region . These findings indicate that mitochondrial fission may be required for in situ proliferation of astrocytes in the CA1 region. Taken together, our findings suggest that the mitochondrial dynamics may play an important role in determining the cell death patterns as well as reactive gliosis of astrocytes in the CA1 region following SE.In the CA1 region, Mdivi-1 increased mitochondrial length (2.97 μm), but WY14643 decreased it in astrocytes of non-SE animals (0.59 μm; The remaining question is what kinds of mitochondrial dynamics-associated proteins are involved in SE-induced astroglial death. Since OPA1 is required for mitochondria fusion (Chen et al., Unlike OPA1, DRP1 plays an important role in the modulation of mitochondrial fission (Smirnova et al., p < 0.05 vs. non-SE, Figures p < 0.05 vs. non-SE, Figures p < 0.05 vs. non-SE, Figure Three days after SE, pDRP1-S616 intensity was increased to 1.89-fold of non-SE level, while pDRP1-S637 intensity was reduced to 0.66-fold of non-SE level in the molecular layer of the dentate gyrus (p < 0.05 vs. non-SE, Figure p < 0.05 vs. non-SE, Figures p < 0.05 vs. non-SE, Figures p < 0.05 vs. non-SE, Figures p < 0.05 vs. non-SE, Figure Four weeks after SE, pDRP1-S616/pDRP1-S637 ratio was declined to 1.66 in the molecular layer of the dentate gyrus (ex vivo study using brain slices are similar to those in in vivo model (Kim et al., Astrocytes regulate the functions the blood–brain barrier, synaptic transmission and energy metabolism in the brain (Anderson and Swanson, Clasmatodendrosis is an irreversible astroglial damage reported by Alzheimer in 1910, which includes extensive swelling and vacuolization of cell bodies and disintegrated and beaded processes, and termed “clasmatodendrosis” by Cajal. In addition, the degeneration of CA1 astrocytes is observed in various models for neurological diseases (Tomimoto et al., in vivo. However, astroglial response to pilocarpine in ex vivo model is similar to that in in vivo model (Kim et al., per se. In the present study, the morphology of mitochondria was conserved between CA1 astrocytes and astrocytes within the molecular layer of the dentate gyrus. However, DRP1 S616/S637 phosphorylation ratio in CA1 astrocytes was higher than in the molecular layer of the dentate gyrus. These findings indicate the higher potentials of mitochondrial fission and DRP1 activity in CA1 astrocytes than in astrocytes within the dentate gyrus. Interestingly, the present data also demonstrates that SE elongated mitochondrial length in CA1 astrocytes via increase in pDRP1-S637 level, while it increased mitochondrial fission due to enhancement of pDRP1-S616, and concomitantly resulted in astroglial death in the dentate gyrus. Therefore, it is likely that the distinctive DRP1 S616/S637 phosphorylation ratio in naïve astrocytes may represent the differential properties of astrocytes, and may play an important role in the proper maintenance of mitochondrial dynamics in response to mitochondrial division-inducing events.It is well known that the vulnerability of neurons to insults is not uniform in the hippocampus, but is heterogeneous among the various brain regions. Briefly, dentate granule cells are remarkably resistant to neuronal damage caused by most insults. Conversely, the CA1 and CA3 neurons as well as hilar neurons are extremely vulnerable to various harmful stimuli (Greene et al., The present data cannot directly address the mechanism by which SE affects mitochondrial dynamics in astrocytes. Horn et al.  reportedIn conclusion, the present findings indicate that naïve astrocytes display unique patterns of DRP1 phosphorylation in the different hippocampal regions, which is relevant to the distinct astroglial responses to SE. In addition, our findings represent that enhanced pDRP1-S637 promoted autophagic astroglial death, while increased pDRP-S616 could induce apoptotic astroglial death. Therefore, the regulation of DRP1 phosphorylation may be one of the important factors to determine the patterns of astroglial death induced by SE.J-EK designed and supervised the project. J-EK designed and performed the experiments described in the manuscript with A-RK, H-WH and S-JM. H-WH, A-RK and J-EK analyzed the data. J-EK wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PD = 125), 3) a-tPCS with 250 ms pulse duration (a-tPCSPD = 250), 4) a-tPCS with 500 ms pulse duration (a-tPCSPD = 500) and 5) sham a-tPCS. The total charges were kept constant in all experimental conditions except sham condition. Post-hoc comparisons indicated that a-tPCSPD = 500 produced larger CSE compared to a-tPCSPD = 125 (P<0.0001), a-tPCSPD = 250 (P = 0.009) and a-tDCS (P = 0.008). Also, there was no significant difference between a-tPCSPD = 250 and a-tDCS on CSE changes (P>0.05). All conditions except a-tPCSPD = 125 showed a significant difference to the sham group (P<0.006). All participants tolerated the applied currents. It could be concluded that a-tPCS with a PD of 500ms induces largest CSE changes, however further studies are required to identify optimal values.The aim is to investigate the effects of pulse duration (PD) on the modulatory effects of transcranial pulsed current (tPCS) on corticospinal excitability (CSE). CSE of the dominant primary motor cortex (M1) of right first dorsal interosseous muscle was assessed by motor evoked potentials, before, immediately, 10, 20 and 30 minutes after application of five experimental conditions: 1) anodal transcranial direct current stimulation (a-tDCS), 2) a-tPCS with 125 ms pulse duartion (a-tPCS Non-invasive induction of neuroplastic changes by transcranial stimulation techniques have been increasingly used in recent years. Transcranial direct current stimulation (tDCS) is a well-known neuromodulatory technique in which the direction of corticospinal excitability (CSE) changes depends on the polarity of the active electrode . LiteratIn a recent study Jaberzadeh et al. (2014) demonstrated that conversion of direct current into unidirectional pulsatile current increases its efficacy for enhancement of CSE. The new paradigm was called transcranial pulsed current stimulation (tPCS).In this paradigm, the direct current was interrupted to take advantage of two extra parameters, “pulse duration” (PD) and “inter-pulse interval (IPI)” . CompareTranscranial alternating current stimulation (tACS) is another neuromodulatory paradigm which has been introduced to modulate human cortical excitability –9. This While the physiological mechanisms behind the effects of tPCS are not well understood, it was assumed that tPCS also induces its effects not only by polarity-dependent modulation of the baseline activity of the neurons in the primary motor cortex (M1), but also through the on–off effects of the pulses on M1 neurons. The introduction of tPCS raises a number of interesting and important questions about the role of these parameters in induction of CSE changes. For example it is unclear what is the optimal PD or IPI for induction of maximum CSE changes in M1 or which combination of PD or IPI induces maximum CSE changes.Our previous research  indicateEleven healthy participants,  with mean age 33.2 ± 7.21 (range 20–51 years) participated in this study. The sample size with a power of 80% was calculated based on a pilot study with 6 participants. All partPD = 125ms, 3. a-tPCS PD = 250ms, 4. a-tPCS PD = 500ms, and 5. Sham a-tPCS. Participants were blinded to the different stimulation conditions. The order in which the experimental conditions were conducted was randomized between participants.A single-blinded randomized sham-controlled cross-over design with 72 hours of washout period was used in this study. Participants were tested before, immediately after, and every 10 minutes up to 30 minutes after the end of 5 different experimental conditions : 1. a-tD2) was placed over the left M1 for the right first dorsal interosseous (FDI) muscle as identified by TMS, and the cathode electrode (35 cm2) was placed over the right contralateral supraorbital area [2). The total charge was calculated based on the following formula:2), SI is the stimulus intensity , ES is electrode size (cm2) and TSD is the total stimulation duration.An Intelect Advanced Therapy System  was used to deliver a-tDCS through a pair of saline-soaked surface sponge electrodes. The active electrode  was placed over the left M1 for the right FDI muscle as identified by TMS, and the cathode (35 cm2) was placed over the right contralateral supraorbital area [PD = 125: current intensity: 1.5 mA, pulse duration: 125 ms, IPI: 50 ms and with a total duration of 20 minutes . For a-tPCSPD = 250 it was: current intensity: 1.5 mA, pulse duration: 250 ms, IPI: 50 ms and with total duration of 10 minutes  and for a-tPCSPD = 500 it was: current intensity: 1.5 mA, pulse duration: 500 ms, IPI: 50 ms and with total duration of 5 minutes . The total charge for a-tPCS applications were calculated based on the following formula:2), SI is the stimulus intensity , ES is the electrode size (cm2), PD is the pulse duration (mSec) and NP is the number of pulses.The same device was used for delivery of a-tPCS through a pair of saline-soaked surface sponge electrodes. The anode  was terminated after 30 seconds of stimulation. Hence participants felt initial sensations, but received no current for the remainder of the stimulation period (9 minutes and 30 seconds mins). This procedure permitted the blinding of participants for the respective stimulation condition [For the sham stimulation, the electrodes were placed in the same positions as for experimental conditions 1–4; however, delivery of a-tPCS  stimulator with a flat 70 mm figure-of-eight magnetic coil. Using the international 10–20 system, the vertex (Cz) point was measured and marked to be used as a reference [Participants were seated upright and comfortable with their head and neck supported by a headrest. Single-pulse magnetic stimuli were carried using a Magstim 200eference . The mageference –20 from Participants were seated in an adjustable podiatry chair with their forearm pronated and the wrist joint in a neutral position, resting on the armrest of the chair. To ensure good surface contact and reduce skin resistance, a standard skin preparation procedure of cleaning and abrading was performed for each site of electrode placement , 21, 22.In this study, 15 MEPs were averaged off-line  for eachBaseline MEP amplitudes and RMT of the respective conditions were tested using one-way repeated measure ANOVA to see whether the baseline MEPs or RMT were identical in all conditions. This test was also carried-out on the mean values from NAS test to analyze the perceived sensation differences between different conditions.P < 0.05 for all statistical analyses. Bonferroni corrections were used as appropriate. All results are presented as the mean ± standard error of mean (SEM) and statistical analyses were performed using SPSS software version 20.A two-way repeated measure ANOVA was used to evaluate the effects of different current types on MEPs’ amplitude over time. The first within-subject independent factor was “current types” with five levels. The second independent factor was “time” with five levels. Mauchly's sphericity test was used to validate an assumption of repeated measures factor ANOVA. Greenhouse-Geisser corrected significance values were used when sphericity was lacking. Post-hoc comparisons were performed using the Bonferroni adjustment for multiple comparisons when appropriate. As an additional analysis, it was assessed whether sham and active stimulations were distinguishable. The participants were asked to indicate if they thought the stimulation was either active or sham at the end of their participation in all testing conditions. Data were analyzed using Pearson’s chi-square. The results were considered significant at the level of PD = 125, a-tPCSPD = 250, a-tPCSPD = 500 and sham a-tPCS in a frequency dependent manner. There were no side effects reported by participants after the conclusion of experimental sessions. There were no reports of burning sensations, pain or headaches during or after a-tDCS or a-tPCS applications. The results of Bonferroni post-hoc comparisons for sensations are listed in All participants tolerated the applied currents in different experimental conditions with no interruption of experimental procedures due to adverse or side effects of the applied currents. 2 (8df) = 5.66, P = 0.68), suggesting that participants could not accurately verify the type of stimulation received and they were successfully blinded. Overall, the percentage of participants who correctly guessed the active condition was 27.2% (excluding ‘cannot say’ responders) and 81.8% (including ‘cannot say’ responders). = 1.68, P = 0.32, partial ŋ2 = 0.18) and RMT  = 1.41, P = 0.36, partial ŋ2 = 0.19) were identical for all conditions.One-way repeated measure ANOVA showed that baseline MEPs’ amplitude (FP = 0.078), current type  and current type × time interaction .The assumption of sphericity had been met for time , time  = 38.02, P < 0.001, partial ŋ2 = 0.79) and the interaction of current type × time  = 6.99, P < 0.001, partial ŋ2 = 0.41)  = 36.009,  = 0.41)  Fig 3)..P < 0.00PD = 500 produced larger increases in CSE compared to a-tPCSPD = 125 , a-tPCSPD = 250  and a-tDCS . However, there was no significant difference between a-tPCSPD = 250 and a-tDCS on CSE changes (P > 0.05)  and sham a-tDCS  respectively. The results also showed that there was no significant differences between a-tPCSPD = 125 and sham tPCS on CSE changes (P > 0.05). However, all other three current types  showed significant changes of CSE compared to the sham stimulation (P < 0.001).Post-hoc comparisons for the main effect of the “Current type” indicated that a-tPCS > 0.05) . a-tPCSPPD = 250, and a-tPCSPD = 500 resulted in significant excitability enhancement lasting at least for 30 minutes (P < 0.005). However, following both sham a-tPCS and a-tPCSPD = 125, there were no significant changes in the MEPs amplitude in different time points (P > 0.05).The lasting effects of changes in different conditions are presented in A paired sample t-test with Bonferroni correction was used to compare mean MEPs in different time points between different conditions . The staIn general, the current study supports the use of a-tPCS, with minimal or no side effects, in healthy participants. In line with the findings of Jaberzadeh et al. (2014), the participants tolerated a-tPCS better than conventional a-tDCS. No adverse effects were recorded or resulted in termination of the experiments. The reported side effects in the current study are consistent with ones reported earlier and include tingling and itchiness sensations . These sIn line with the findings of Jaberzadeh et al. (2014), application of a-tPCS, induced phosphene (light flashing) in 54% of participants’ eyes. The rate of these light flashings was correlated to the frequency of the applied pulses , 31. ThiPD = 500 and PD = 250). On the contrary, a-tPCS with shorter PD (PD = 125) failed to show any post intervention increase in CSE. Current knowledge on tPCS effects on the CSE changes is limited, and the mechanisms underpinning this effect are not yet understood. Different mechanisms may explain the results presented in this study. It is already established that the effect of a-tDCS is due to unchanged flow of direct current [tonic and phasic effects of the applied pulses .Firstly, it was hypothesized that both a-tDCS and a-tPCS of M1 would induce an increased CSE, which would remain significant up to 30 minutes post intervention. This hypothesis was partially supported by the findings in the present study. This increase was only found following a-tDCS and a-tPCS with longer PDs ( current . Convers2) constant, the application time of a-tPCS and therefore the number of pulses per application were different. The application times of a- tPCS were 20, 10 and 5 minutes for PDs of 125 and 250 and 500 milliseconds, respectively. Interestingly, it was found that shorter application of a-tPCS, as in a-tPCSPD500, induced larger CSE changes than longer a-tPCS applications. The main question is how the prolonged stimulation period in PD-125 and PD-250 might contribute to the absence/weaker effect in comparison to other a-tPCS conditions? In exploring this question it should be noted that longer applications of a-tPCS, as in PD-125 and PD-250, apply lower NDCC , intensity (1.5 mA) and total charge (~ 17 mC/ cmwer NDCC . This inPD = 500). While a-tPCS PD = 125 failed to induce any CSE changes, the a-tPCS PD = 250 only induced CSE changes comparable to a-tDCS   and GropThe rationale behind os-tDCS or tACS protocols is to interact with endogenous oscillatory cortical activity. In alignment with the findings of the current study, Bergmann  and GropThe third hypothesis in the current study indicated that a-tPCS of M1 does not have any sham effects. The results of the present study support this hypothesis. This finding is in line with the finding of our previous a-tPCS study , which iThe findings in the current study must be considered in the context of the following limitations. Firstly, even though it has been shown that the induction of retinal phosphene on its own does not have any effect on CSE , its intThis study evaluated the effects of three different PDs on the size of induced CSE in M1. Further investigation is required to identify the optimal PD of tPCS for generation of M1 CSE changes. The current study evaluated the lasting effects of a-tPCS over a time duration of up to 30 min. Further studies are required to extend the lasting effects of a-tPCS when applied for longer duration. The effects of different characteristics of pulsatile currents such as amplitude and frequency of the pulses, are additional areas which should be scientifically explored to define the optimal parameters for prolongation of tPCS lasting effects. In addition to cortical changes, future studies should also assess behavioural changes such as motor performance in both healthy individuals and patients with neurological conditions. Furthermore, to underline the mechanisms behind efficacy of a-tPCS, a mechanistic study of motor cortex excitability should be undertaken, by measuring silent period, intracortical inhibition, and facilitation, to assess the function of inhibitory and facilitatory neurotransmitters. Additional experiments including simultaneous assessment of EEG are required to refine tPCS as a method of frequency dependent brain stimulation.


Human papillomavirus (HPV) self-sampling (self-HPV) is valuable in cervical cancer screening. HPV testing is usually performed on physician-collected cervical smears stored in liquid-based medium. Dry filters and swabs are an alternative. We evaluated the adequacy of self-HPV using two dry storage and transport devices, the FTA cartridge and swab.A total of 130 women performed two consecutive self-HPV samples. Randomization determined which of the two tests was performed first: self-HPV using dry swabs (s-DRY) or vaginal specimen collection using a cytobrush applied to an FTA cartridge (s-FTA). After self-HPV, a physician collected a cervical sample using liquid-based medium (Dr-WET). HPV types were identified by real-time PCR. Agreement between collection methods was measured using the kappa statistic.HPV prevalence for high-risk types was 62.3% (95%CI: 53.7–70.2) detected by s-DRY, 56.2% (95%CI: 47.6–64.4) by Dr-WET, and 54.6% (95%CI: 46.1–62.9) by s-FTA. There was overall agreement of 70.8% between s-FTA and s-DRY samples (kappa = 0.34), and of 82.3% between self-HPV and Dr-WET samples (kappa = 0.56). Detection sensitivities for low-grade squamous intraepithelial lesion or worse (LSIL+) were: 64.0% (95%CI: 44.5–79.8) for s-FTA, 84.6% (95%CI: 66.5–93.9) for s-DRY, and 76.9% (95%CI: 58.0–89.0) for Dr-WET. The preferred self-collection method among patients was s-DRY (40.8% vs. 15.4%). Regarding costs, FTA card was five times more expensive than the swab (~5 US dollars (USD)/per card vs. ~1 USD/per swab).Self-HPV using dry swabs is sensitive for detecting LSIL+ and less expensive than s-FTA.43310942International Standard Randomized Controlled Trial Number (ISRCTN):  The burden of cervical cancer (CC) remains very significant, especially in developing countries, and accounts for around a quarter of a million deaths annually worldwide . In the Human papillomavirus (HPV) testing appears to overcome some inherent barriers of cytological screening. Detection sensitivity for cervical intraepithelial neoplasia grade 2 or worse (CIN 2+) of HPV testing is twice that of cytology . FurtherA great variety of collection devices has been used in studies on self-sampling for HPV-testing (self-HPV). The most common devices are tampons, swabs, cervicovaginal brushes, and cervicovaginal lavage. In current practice, samples for HPV testing are stored in a liquid-based medium, which requires careful handling owing to its flammability and toxicity. The need for stable transport and storage temperatures makes such testing methods difficult and costly to provide in developing countries. Furthermore, despite women’s high acceptance of self-HPV, they remain concerned about the validity of the method and are afraid of spilling the transport medium during the sampling procedure and transport , 13–15.Dry storage and transport might be a valuable option. The FTA elute cartridge  is a dry carrier that immobilizes and stabilizes nucleic acids from fresh samples. This biohazard-free paper is chemically treated with proprietary reagents that lyse cells upon contact, denaturing proteins. The FTA cartridge contains an indicating dye that changes color when a sample is applied, thereby confirming that the procedure has been performed properly. Moreover, easy storage and transport at room temperature is possible.Evidence shows good agreement for detection of HPV DNA between samples collected in the FTA cartridge (s-FTA) and in a liquid-based medium , 15–19. Alternatively, vaginal dry swabs (s-DRY) are inexpensive and are not usually associated with a great loss of cellular material for analysis. Studies have shown that self-HPV swabs can be successfully transported in a dry state at ambient temperature without compromising specimen integrity, and that there is good agreement (70–90%) for HPV detection between dry and wet swabs , 21.Although the feasibility of both the FTA cartridge and s-DRY as self-collection methods for HPV detection has been compared in several studies to standard swab collection in a liquid medium, to our knowledge, the relationship between performance of the FTA cartridge and s-DRY for HPV detection has never been addressed. Our goal was to evaluate the acceptability and analytic performance of two dry storage and transportation devices, s-FTA and s-DRY, and comparing them with the current standard of HPV testing using physician-collected samples and a specimen transport medium (Dr-WET).From March 2014 through February 2015, we enrolled women from the colposcopy clinic of Geneva University Hospitals, Switzerland. Women were eligible if they were over 30 years old, and if they understood the study procedures and voluntarily agreed to participate by signing a written informed consent form. No follow-up was done. Pregnant women, those with a history of hysterectomy, and who did not consent to participate, were excluded. A sample of 150 women was consecutively recruited (20 women were excluded as they did not meet the inclusion criteria or declined to participate). The Cantonal Human Research Ethics Commission of Geneva (CCER) approved the study .™; COPAN Italia, Brescia, Italy) for HPV testing (Dr-WET). The sequence of the two self-HPV tests was randomized to avoid potential bias that might favor the “first” test. A research nurse gave oral instructions to participants, who were instructed to wash their hands before the specimen collection procedure. Each participant received a package containing a specimen collection kit. The Rovers® Viba-Brush®  was used for self-collection with the FTA cartridge, and the mid-turbinate flocked vaginal swab  used for self-collection with the s-DRY method. Recommendations were to hold the brush or swab by the end of its handle and insert the brush or swab into the vagina, avoiding contact with the external genitalia, until resistance was felt (at least 6 cm). While gently maintaining pressure, participants were instructed to turn the brush or swab three to five times. Subsequently, the brush was to be applied to the FTA cartridge by pressing it onto the middle of the indicated sample area and then rotate the brush three to five times across that area. The FTA elute matrix contains an indicating dye that changes color from purple to white when a sample has been applied correctly. Three small circles of approximately 3.0 mm diameter were cut from the center section of the FTA card with a disposable sterile carbon steel surgical blade, and placed into a 1.5-ml microcentrifuge tube. A new sterile blade was used for each card.All eligible women were invited to collect two self-samples (s-FTA and s-DRY). A physician subsequently collected a third cervical sample with a swab immersed in a collection medium  was simply inserted into its plastic sleeve for storage and transport. During the subsequent colposcopy consultation, a physician also collected a sample for HPV testing.After all specimen collection, participants completed a self-administered questionnaire to collect sociodemographic data and query acceptance of the test. The FTA-card punches, flocked swabs, and ESwabs were immediately forwarded to the laboratory for analysis.This trial was registered at ISRCTN Registry as ISRCTN43310942 . The study protocol is available in S2 Text.Elution from FTA card: The 3.0-mm FTA punch discs were washed in 500 μl of sterile water and pulse vortexed three times for 5 s. The water was removed from each tube and the punch discs were transferred to a new microfuge tube containing 200 μl distilled water, which was then transferred to a heating block at 95°C for 30 min. The sample was then removed, pulse vortexed three times for 5 s, and centrifuged for 30 s to separate the matrix from the eluate. The eluted material was transferred to a new tube and stored at –20°C until DNA extraction.Material recovery from dry swab sample (s-DRY): Each sample was placed into 1 ml of sterile phosphate-buffered saline (PBS), and each tube was pulse vortexed three times for 15 s before removing and discarding the swab collection device. Samples were then stored at −20°C until extraction.Material recovery from ESwab sample (Dr-WET): Tubes containing the Dr-WET sample were also pulse vortexed three times for 15 s, and then the swab was discarded. Samples were then stored at −20°C until automated extraction.DNA extraction: The entire volume of recovered material was transferred into appropriate tubes and the volume adjusted to 600 μl with PBS. DNA was then extracted on an m2000sp instrument , according to manufacturer instructions. The DNA elution volume was 100 μl/sample.HPV and genotyping: HPV analyses were performed using the Anyplex II HPV28 (H28) Detection test . The H28 is a semi-quantitative real-time multiplex PCR 30 assay for screening and genotyping 28 HPV genotypes [™) and Tagging Oligonucleotide Cleavage and Extension (TOCE™) technologies, allowing simultaneous detection of 19 high-risk HPVs  and nine low-risk HPVs. The 19 high-risk HPVs detected in the test include HPV types defined by the International Agency for Research on Cancer as Group 1 (carcinogenic to humans), Group 2A (probably carcinogenic to humans), and Group 2B (possibly carcinogenic to humans). Analysis was done as recommended by the manufacturer.enotypes . This teA sample size of 130 women was needed to validate differences in test performance of 10% or more, assuming a 40–50% prevalence of HPV infection in the selected population. Agreement between the collection methods according to cytological results was measured using the kappa statistic (ĸ) and corresponding standard deviation (SD). The proportion of positive agreement (PPA) between paired s-FTA and s-DRY samples was calculated using 2a/(f1 + g1), where a is the number of samples that were positive for HPV in both dry samples, f1 is the number of samples that were positive for s-FTA, and g1 is the number of samples that were positive for s-DRY. The same was done for self-collection methods (combined results of s-FTA and s-DRY) and Dr-WET.Sensitivity and specificity for detection of high-risk HPV using Dr-WET as the gold standard was reported, as well as sensitivity and specificity of the three sampling methods for abnormal Pap smear. Because of the small number of high-grade squamous intraepithelial lesions (HSIL) or carcinomas in our population, we assessed sensitivity and specificity for low-grade squamous intraepithelial lesion or worse (LSIL+). The two-tailed McNemar’s test was used for mutual comparison of sensitivity and specificity. Positive and negative predictive values were also calculated. Data were analyzed using Stata Statistical Software Release 13 .One hundred and thirty patients were included in the study. Study flowchart is described in HPV prevalence for high-risk types was 62.3% (95% CI: 53.7–70.2) detected by s-DRY, 56.2% (95% CI: 47.6–64.4) by Dr-WET, and 54.6% (95% CI: 46.1–62.9) by s-FTA. Infection with HPV types 16 and 18 alone was identified in 4.6% of participants, and infection together with other high-risk types in 13.8% of cases. Infection with high-risk genotypes other than types 16 and 18 was found in 50% of women.The distribution of HPV genotypes is shown in Cytological results obtained in the 12 months prior to the patient recruitment date or during the study were considered in the data analysis Tables  and 5. WOverall agreement between self-HPV and physician-collected samples when LSIL+ was present was 89.7% (ĸ = 0.61), and the PPA was 93.0%. Overall agreement between FTA cards and Dr-WET samples was 83.1%, and kappa was 0.64 (0.50–0.77). Overall agreement between s-DRY and Dr-WET samples was 85.2%, and the kappa was 0.64 (0.50–0.78)Agreement between the s-FTA versus Dr-WET, s-DRY versus Dr-WET, and s-FTA versus s-DRY for the number of oncogenic HPV types is shown in Clinical performance of self-collection and physician-collection methods using the Dr-WET sample results or the cytological results (LSIL+) as gold standard is represented in Sample preferences and opinion about the collection methods are represented in To our knowledge, this is one of the first studies to compare acceptability and analytic performance between two dry storage and transportation devices for HPV detection, the FTA cartridge and dry swab.Overall, detection of HPV was significantly less common with the s-FTA method relative to s-DRY or Dr-WET, using the same test for HPV detection (Anyplex II HPV28). This finding is consistent with a previous trial in Barcelona , even when a real-time PCR method was used for HPV testing in both collection methods. Indeed, previous studies have demonstrated that s-FTA is a favorable method for HPV diagnosis in the context of GP5/6-PCR use, but not Hybrid Capture 2 , 18. In Overall, several studies have shown substantial agreement for PCR HPV detection and genotyping between cervical cells collected using a liquid-based medium and specimens collected using FTA cartridges, among both self-collected and clinician-collected samples , 18, 23.This study has some limitations that must be addressed. The self-collected samples were always done before the physician-collected sample, creating a bias in favor of the self-HPV. Another limitation is the uncertainty that our findings can be generalized to other populations with low HPV prevalence; our study was based on adult women in a colposcopy clinic with a high prevalence of HPV infection.The strengths of our study include the possibility for comparing the s-FTA, s-DRY and Dr-WET methods within categories of cytological diagnoses and, more specifically, for evaluating the performance of s-FTA and s-DRY among women with an LSIL+ diagnosis. Furthermore, by randomizing the sequence of the two self-collection methods, we did not favor one method over the other.™ Elute Micro Card) with the dry swab (COPAN FLOQSwabs™), we found that the card is five times more expensive (~5 USD per card vs. ~1 USD per swab). This does not even consider the price of the entire kit, which includes the brush for collection and puncher for extraction. In addition to these disadvantages, the method would be difficult to implement in a context of low human and material resources, owing to laborious sample processing. In the present study, testing was done according to strict standard laboratory procedures to avoid PCR contamination. Punching of the FTA card is not only expensive, it is labor-intensive, and cross-contamination can readily occur if rigorous techniques are not used. In this study, a disposable surgical blade was used and we have no reason to believe that this would affect card performance, as this technique has been previously used without compromising the results [Although the FTA card may have some advantages such as the assurance of correct test performance by means of the color indicator, it is not as sensitive as other collection methods and has a higher cost. Indeed, by comparing the regular prices of the card Click here for additional data file.S1 TREND Checklist(PDF)Click here for additional data file.


Streptococcus mitis (S. mitis) and Pseudomonas aeruginosa (P. aeruginosa) are typically found in the upper respiratory tract of infants. We previously found that P. aeruginosa and S. mitis were two of the most common bacteria in biofilms on newborns’ endotracheal tubes (ETTs) and in their sputa and that S. mitis was able to produce autoinducer-2 (AI-2), whereas P. aeruginosa was not. Recently, we also found that exogenous AI-2 and S. mitis could influence the behaviors of P. aeruginosa. We hypothesized that S. mitis contributes to this interspecies interaction and that inhibition of AI-2 could result in inhibition of these effects. To test this hypothesis, we selected PAO1 as a representative model strain of P. aeruginosa and evaluated the effect of S. mitis as well as an AI-2 analog (D-ribose) on mono- and co-culture biofilms in both in vitro and in vivo models. In this context, S. mitis promoted PAO1 biofilm formation and pathogenicity. Dual-species (PAO1 and S. mitis) biofilms exhibited higher expression of quorum sensing genes than single-species (PAO1) biofilms did. Additionally, ETTs covered in dual-species biofilms increased the mortality rate and aggravated lung infection compared with ETTs covered in mono-species biofilms in an endotracheal intubation rat model, all of which was inhibited by D-ribose. Our results demonstrated that S. mitis AI-2 plays an important role in interspecies interactions with PAO1 and may be a target for inhibition of biofilm formation and infection in ventilator-associated pneumonia. Interactions among diverse microbial species are dynamic and most likely propel many adaptations, such as biofilm formation in host respiratory tracts and multiple marine habitats . These iStreptococcus mitis (S. mitis) and Pseudomonas aeruginosa (P. aeruginosa) were two of the most common bacteria in biofilms on newborns’ ETTs and in their sputa (S. mitis could influence the behaviors of P. aeruginosa (Ventilator-associated pneumonia (VAP) is one of the most common acquired infections in intensive care units (ICUs), occurring in 10–25% of mechanically ventilated patients . The preir sputa . Recentlruginosa .Streptococcus mitis is one of the predominant bacteria that colonize the upper respiratory tract of newborns and is generally considered to be harmless  on mono- and co-culture biofilms in vitro and in an endotracheal intubation rat model. We focused on the influence of S. mitis and D-ribose on PAO1 and specifically evaluated the effects on biofilm formation, virulence and the expression of PAO1 QS genes. This work may provide important insight into VAP caused by multibacterial infection, and particularly neonatal VAP.In the present study, we aimed to explore the effects of S. mitis (ATCC 49456) and PAO1. The PAO1 was kindly provided by Mr. Zhijin Chen  was measured at 600 nm at intervals of 2 h for up to 24 h using a spectrophotometer . All experiments were performed three times independently , and 20 μl of cell-free culture supernatant or synthesized AI-2 was added to 180 μl of diluted V. harveyi cells. Cell-free culture medium from V. harveyi BB170 was included as a positive control, and sterile BHI medium and PBS were included as negative controls. The reaction was carried out at 30°C, and light production was monitored using a Thermo Scientific Fluoroskan Ascent FL .Testing for AI-2 was previously described . Briefly600A = 0.05) of PAO1  and S. mitis  were combined in a 96-well transwell microtiter plate (Corning). BHI broth (150 μl) containing PAO1 (50 μl), BHI broth (50 μl) containing S. mitis (150 μl), and BHI broth (200 μl) alone served as three different control groups. We previously determined that the optimal ratio of the bacterial species is 1:3 (PAO1:S. mitis) (data not shown). Then, 22 μl of D-ribose (500 μM) or sterilized ddH2O was added to each well. The plates were subsequently incubated at 37°C in a 5% CO2, humidified atmosphere for 24 h.To study the direct interactions between the species, standardized suspensions  and resuspended in 100% methanol for 2 h. Dead cells were then centrifuged and washed three times with PBS to remove any remaining trace of methanol. Finally, the dead cells were resuspended to 600A = 0.05. To confirm bacterial killing, aliquots of the bacterial cells were spread onto blood agar plates and incubated overnight at 37°C and 5% CO2. A total of 100 μl of the standardized suspension of PAO1 was combined with methanol-killed bacterial cells (PAO1 or S. mitis) in a 96-well microtiter plate and incubated for 24 h at 37°C and 5% CO2. The biomass was then quantified by crystal violet assay.To study the indirect interactions between the species, we employed co-cultivation of PAO1 with supernatant from eviously . BrieflyA570 of the solubilized dye was obtained using an Epoch microplate spectrophotometer (BioTek).The biofilm biomass was assessed using a modified version of a protocol first developed by A600 = 0.05) of PAO1 in the lower compartment and 0.75 ml of BHI broth or S. mitis (A600 = 0.05) in the upper compartment. Half of the PAO1&S. mitis wells also received D-ribose at a final concentration of 50 mM. After 24 h of incubation at 37°C and 5% CO2, the liquid medium was removed. The coverslips were then gently washed with PBS, and the biofilm mass, cell number, and structure were analyzed using microscopic techniques (see below).For transwell studies, uncoated 24-well transwell polystyrene cell culture plates (Corning) with one coverslip per well were inoculated with 0.25 ml (For scanning electron microscopy (SEM) studies, an inoculum was prepared as previously described . For conIn parallel experiments, samples were prepared for RNA extraction and viability counts. Viable cell numbers were obtained from biofilms on coverslips in 24-well polystyrene cell culture plates after two gentle washes with PBS. The resulting suspensions were serially diluted in PBS, and 50 μl aliquots were plated on Columbia sheep blood agar. Colony-forming units (CFU) were counted after 16–18 h of incubation, and the distinct colony morphologies allowed for differentiation between the two species.Total RNA was extracted from the bacteria adhering to the coverslips using the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa Bio). Reverse transcription was then performed using the TaKaRa PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa Bio) according to the manufacturer’s instructions. cDNA samples from the same biological sample were pooled, diluted six times and stored at –20°C until use.2O were added to the reaction wells. A no-template control (NTC) was included during each Q-PCR experiment to check the purity of the reagents. Each reaction was performed in duplicate, and the experiments were repeated four times, with different RNA samples used each time.SsoFast EvaGreen Supermix (Bio-Rad) was used for real-time amplification and for visualization of the amplified cDNA. In particular, 1 μl of a cDNA dilution, 0.4 μl each of forward and reverse primers, 5 μl of SsoFast EvaGreen Supermix, and 3.2 μl of sterile HTable 1. A single sharp peak and a single band of the expected size were observed in the melting curve and in the agarose gel, respectively. The identities of the products were confirmed by DNA sequencing.The Q-PCR reactions were performed in a CFX96 Real-Time PCR Detection System (Bio-Rad). The thermal cycling conditions started with 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 5 s. The primer sets used for these analyses are listed in tC method, and the expression levels of the same genes in mono- and co-cultures of PAO1 and S. mitis were compared. In this study, the expression of target genes in mono-cultures acted as the control. The tC values of both the control and the genes in question were then normalized to that of the PAO1 housekeeping gene (rpsL) (Whether the expression of certain target genes in PAO1 could be reproducibly induced or affected by systematic co-culturing under multi-strain growth conditions was investigated. Relative mRNA expression was quantified using the efficiency-corrected comparative e (rpsL) .tΔΔC analysis) was applied to evaluate the relative changes in gene expression in the Q-PCR experiments. The computer programs GenEx (Bio-Rad) and Excel (Microsoft) were used to solve the following equation: ΔΔCt = ΔΔCt, sample–ΔΔCt,reference (Bio-Rad)  .S. mitis four times, whereas the other tubes were washed 3 times. These different protocols were used because the mixed PAO1&S. mitis biofilm contained more bacteria than the PAO1 biofilm did. The CFU value of the PAO1 biofilm and the mixed PAO1&S. mitis biofilm after 72 h of incubation and before endotracheal intubation was approximately 1 × 107, whereas the CFU value of the S. mitis biofilm was less than ∼104.To assess how lung infection changes between ETTs covered in single- and dual-species biofilms, an endotracheal intubation rat model was used. For this purpose, disposable sterile plastic scalp acupuncture tubes of 3.0 mm in diameter were cut to 1 cm in length and precoated with bacteria before endotracheal intubation. The prepared tubes were specifically immersed in the same bacterial suspension as that used in the transwell biofilm assay described above. After biofilms formed on the surface of the inoculation tubes, to estimate the bacterial count in these biofilms, the bacteria were detached from the tubes by consecutive ultrasonic shaking and vortexing for 2 min and 5 min, respectively. Our preliminary results indicated that this process did not affect cell culturability. To make the density of bacteria in each group approximately equivalent, we washed the tubes covered in both PAO1 and S. mitis (PS group), PAO1&S. mitis&D-ribose (PSD group), and S. mitis (S group) biofilm-covered tubes were chosen as the four infection groups, and intubation with sterile tubes served as the control. The PSD group was treated with ultrasonic atomization-based inhalation of D-ribose  for 30 min every day, whereas the other groups were treated with ultrasonic atomization-based inhalation of PBS.Sixty adult female Sprague–Dawley (SD) rats, each weighing 200–220 g, were randomly allocated to 4 groups with 15 rats each. Intubations with PAO1 (P group), PAO1&The animals were purchased from the Chongqing Medical University Laboratory Animals Center . All rats were housed in a pathogen-free environment and received sterile food and water in the Laboratory Animal Center at the Children’s Hospital of Chongqing Medical University. The procedure of endotracheal intubation was previously described . Animal 2. The colonies on the plates were subsequently counted to estimate CFU values. Simultaneously, the right lung was lavaged with 3 ml of PBS; this fluid was instilled and withdrawn three times. Following the lavage, the total cell count of the bronchoalveolar lavage fluid (BALF) was measured using a cell counter . The protein levels of IL-4, IL-10, and IL-6 were also measured using commercially available ELISA kits  according to the manufacturer’s instructions. The BALF samples were assayed in duplicate.Half of each left lung was homogenized and prepared for bacterial counting, as described previously . The remt-statistic was used to determine other significant differences between two groups, and a P-value of <0.05 was considered statistically significant. Moreover, comparisons of mortality from infection were performed using Fisher’s exact test.All experiments were performed in triplicate unless stated otherwise. Statistical analyses were performed using SPSS 19 software. The data are presented as the mean ± SD. The D-ribose on planktonic bacterial growth. The 50 mM concentration of D-ribose did not influence the growth of the planktonic PAO1 cultures, whereas it induced the growth of S. mitis in the stationary phase .We first investigated the effect of S. mitis, PAO1 and BB170 after 14 h of growth were screened for AI-2-like activities. AI-2 was observed in the S group and BB170 group, whereas it was not observed in the P group .Cell-free supernatants harvested from representative strains of S. mitis. As shown in Figure 1, the PAO1 strain formed flat, tight biofilms with little heterogeneity and few viable cells, whereas S. mitis alone did not form biofilms. When the two strains were co-cultured , the biofilms had a denser, thicker structure. Based on analysis using COMSTAT software, PAO1&S. mitis biofilms showed significant structural differences compared with the P group and the PSD group. As is shown in Table 3, the biomass, the average thickness, the maximum thickness and the surface area of the biomass all increased in the PS group, whereas the roughness coefficient showed the opposite trend. Interestingly, D-ribose (50 mM) significantly inhibited co-culture biofilm formation (Table 3), whereas there was no significant effect on PAO1 viability or biofilm formation .To confirm whether the reciprocal interactions between the two species could affect their capacity to produce biofilms, we first investigated mono-species biofilms formed by PAO1 or S. mitis could not form biofilms . Co-cultivation of PAO1 with S. mitis resulted in an increase in the total biomass by 41.18% compared with PAO1 mono-species culture, whereas adding D-ribose showed the opposite trend . The addition of ethanol-treated S. mitis cells did not have this effect on PAO1 cultures . The total bacterial colony counts for the PS group also increased by 28.78% compared with the counts in PAO1 biofilms . In addition, S. mitis accounted for approximately 8% of the dual-species biofilms (data not shown). Exposure to S. mitis supernatant also triggered PAO1 adhesion, increasing the biofilm biomass by 30.77%. Heat-treating the supernatant did not significantly reduce this effect, as the biofilm biomass still increased by 22.25% .Under co-culture conditions, we noticed that the biomass and the total number of live cells within the biofilms increased. Crystal violet assays revealed that Table 2 presents the normalized results as relative gene expression under the co-culture conditions compared with expression under the mono-culture conditions. Significant differences were observed in the relative levels of the target genes , with significant overexpression under co-culture conditions.S. mitis biofilms could increase the mortality rate and aggravate lung infection and to analyze the effects of D-ribose, we measured the mortality rate, performed bacteriological examinations and measured the total cell count in the BALF in all groups.To determine whether ETTs covered with mixed PAO1&Figure 3A, the mortality rate over the 7 days following intubation was higher when the tubes were covered in mixed PAO1&S. mitis biofilms compared with when the tubes were covered in PAO1 biofilms, and D-ribose reduced this effect. However, the difference was not statistically significant (P > 0.05), which may have been due to the small sample size.As shown in Figures 3 and 4, the PS group exhibited a heavier bacterial burden and more serious pathological changes, and treatment with D-ribose reduced this effect. In contrast, the bacterial cell counts in the lungs of both the control group and the S group were much lower or negative (P < 0.05).Respiratory infection, which was measured based on histological examination and CFU per gram, occurred in all inoculated mice. As shown in Figures 3D–F, the total number of cells and the IL-10 level in the BALF were significantly higher in the PS group than in the other three groups, and the expression of IL-10 decreased following treatment with D-ribose (P < 0.05). However, there was no significant difference in IL-4 between the PS group and the P group. In contrast, the IL-6 level was significantly lower in the PS group than in the P group.As shown in S. mitis and P. aeruginosa were two of the most common bacteria in biofilms on newborns’ ETTs and in their sputa . These results are in agreement with reports that S. mitis increases biofilm formation by dental and periodontal pathogens (D-ribose) can inhibit dual-species biofilm formation  could inhibit these effects.Multi-species biofilms often possess combined metabolic activity that is greater than that of the component species . Palmer athogens , whereasormation . These fS. mitis is mediated not only by direct contact but also by secreted extracellular molecules. This concept is in accordance with previous investigations that examined supernatants from bacterial strains found in the respiratory and gastrointestinal tracts and identified P. aeruginosa culture supernatants as having inhibitory properties , indicating that the release of small, heat-stable molecules was responsible for this increase in biomass.We have shown that the interaction between PAO1 and operties . We founS. mitis enhanced the expression of the PAO1 QS genes, including lasI, lasR, rhlI, and rhlR (Table 2), and that D-ribose (50 mM) inhibited co-culture biofilm formation. The inhibitory effect of D-ribose on biofilm formation is likely attributable to competitive binding of D-ribose with AI-2 for RbsB  can be enhanced by the presence of oral commensal streptococci, and S. mitis has also been shown to potentiate LES virulence factor production in co-culture biofilms (P < 0.05). IL-10 has emerged as a key immunoregulator during infection  ETTs covered in dual-species biofilms increased the mortality rate and aggravated lung infection compared with ETTs covered in mono-species biofilms in an endotracheal intubation rat model; and (3) all of these effects were inhibited by an AI-2 analog (D-ribose). Overall, our results demonstrated that S. mitis AI-2 plays an important role in interspecies interactions with PAO1 and may be a target for inhibition of biofilm formation and infection in VAP.The present study demonstrated that (1) ZW and JYu conceived and designed the study, ZW and QX performed the study, TY, LL, JYa, and HL analyzed and interpreted the data, ZW and YH wrote the manuscript. JYu, YZ, and QL revised the manuscript. All authors read and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P = 0.005, cP = 0.01, OR = 1.787), and the frequency of the GG genotype was higher in AS patients than in controls . However, no significant association was found between the miR-499 rs3746444T>C variant and susceptibility to AS. This is the first study to address the association between the miR-146a rs2910164G>C and miR-499 rs3746444T>C polymorphisms and AS, and it suggests a potential pathogenic factor for AS. Further studies are needed to validate our findings in a larger series, as well as in other ethnic backgrounds.miRNAs are small, non-coding RNAs that regulate the expression of multiple target genes at the post-transcriptional level. Single-nucleotide polymorphisms (SNPs) in miRNA sequences may alter miRNA expression and have been implicated in the pathogenesis of multiple forms of arthritis, including rheumatoid arthritis (RA) and osteoarthritis. The present study explored the association between ankylosing spondylitis (AS) and two single nucleotide polymorphisms (SNPs), miR-146a rs2910164G>C and miR-499 rs3746444T>C, in a Han Chinese population. A case–control study consisting of 102 subjects with AS and 105 healthy controls was designed. The two miRNA SNPs were identified by direct sequencing. Subsequently, their gene and genotype frequencies were compared with healthy controls. A significant difference was observed in the miR-146a rs2910164G>C SNP. The frequency of the G allele was markedly higher in the AS patients than in the healthy controls ( Ankylosing spondylitis (AS) is a common inflammatory rheumatic disease that affects the axial skeleton, causing characteristic inflammatory back pain. It can lead to structural and functional impairments and a decrease in quality of life. Although the pathogenesis of AS remains ambiguous, it is likely that AS is a multifactorial disease, influenced by genetic and environmental factors. It is conceivable that the HLA-B27 gene is the most important risk factor for AS. However, many genes in and outside the MHC, as yet largely undefined, have also been demonstrated to be associated with the development of AS.MicroRNAs  are a group of endogenous, 20–25 nucleotides long non-coding RNAs. Most miRNAs are transcribed by RNA polymerase II, and their upstream regulatory regions contain canonical core promoters and enhancers, regulated by transcription factors . They fuSingle nucleotide polymorphisms (SNPs) are known to be the most common type of genetic variation in the human genome. SNPs located in miRNA regions can alter miRNA expression and/or maturation to affect function in three ways: through the transcription of the primary transcript, through pri-miRNA and pre-miRNA processing, and by affecting miRNA–mRNA interactions . RecentlIt has been reported that miRNA expression could be altered in synovia, peripheral blood mononuclear cells (PBMCs) or T cells from patients with different forms of arthritis, including RA, OA and AS –21. As mIn our research, we hypothesized that miR-146a rs2910164G>C and miR-499 rs3746444T>C polymorphisms may be involved in the occurrence and development of AS. To test this hypothesis, the two SNPs were genotyped in patients with AS and healthy controls. Then, using statistical analysis, we evaluated the roles of these miRNAs in disease predisposition.HLA-B27-positive and ranged in age from 16 to 79 years, with an average age of 34.88 years. The 105 healthy controls, including 74 males and 31 females, were selected from the health examination center of our hospital. Their ages ranged from 17 to 55 years, with an average age of 32.14 years. This study was approved by the ethics committee of The First Affiliated Hospital of Wenzhou Medical University, approval number CR2014-070. Written informed consent was obtained from all participants. In a case of possible compromised capacity to consent, written informed consent was also obtained from a close relative of the participant. If the participant was a minor, both the participant and the next of kin provided written informed consent. This study was conducted according to the principles expressed in the Declaration of Helsinki.In total, 102 AS patients were enrolled in the study from the First Affiliated Hospital of Wenzhou Medical University between March 2013 and April 2014, and they were diagnosed strictly according to the modified New York criteria. The 83 male and 29 female AS patients were all Genomic DNA was extracted from EDTA anticoagulant blood using the DNAfast2000 DNA Extraction Kit  according to the manufacturer’s instructions. The concentration and purity of DNA were measured using ultraviolet (UV) spectrophotometry (Eppendorf). The extracted DNA was detected immediately or stored at -20°C for less than 6 months.A total of 207 individuals were genotyped for the two SNPs (rs2910164 G>C and rs3746444 T>C) by directly sequencing. The primers used for rs2910164 were 5-CACCCACATCAGCC TTCCAG-3 and 5-CTCCAGGTCCTCAAGCCCAC-3, and the primers used for rs3746444 were 5-ACTTCCCTGCCAAATCCC-3 and 5-GTTCCAGACGGTGTCCCA-3. The polymerase chain reaction (PCR) conditions for rs2910164 were 95°C for 3 min followed by 40 cycles of 95°C for 5 s and 60°C for 30 s, and a final 2 min extension at 70°C. The PCR conditions for rs3746444 were 95°C for 3 min, 40 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 15 s, with a final extension at 72°C for 10 min. The PCR products were sent to GENEMIZ for sequencing to detect the SNPs.P<0.05 was considered statistically significant, and all statistical tests were two-sided.Statistical analyses were conducted using SPSS 15.0. The genotype and allele frequencies of the AS patients and healthy controls were compared using the Chi-squared test or Fisher’s exact test. The genotype distribution was evaluated using the asymptotic Pearson’s v2 test to determine whether it met the conditions of Hardy–Weinberg equilibrium (HWE). The power was calculated using PASS11.0 . The Bonferroni method was used to correct the data. P = 0.005, Pc = 0.01, OR = 1.767). However, the miR-499 rs3746444 T/C SNP showed no association with the susceptibility of AS at the allele level .In total, 102 AS patients and 105 healthy individuals were successfully genotyped for their rs2910164 G/C and rs3746444 T/C SNPs. As shown in P>0.05, data not shown). There was no population stratification and no sampling bias. As shown in P = 0.014, Pc = 0.042, OR = 2.516). However, no difference was observed in the distribution of miR-499 rs3746444 T>C genotypes between patients with AS and controls .The distributions of the genotype frequencies for the two SNPs complied with Hardy-Weinberg equilibrium in both the cases and controls  ,33,34. ARegarding miR-499, although there is no literature directly focusing on the role of miR-499 in the pathogenesis and development of AS, it can be speculated that a certain mutuality does exist. On the one hand, it has been demonstrated that IL-17RB, IL-23a, IL-2RB, IL-6, IL-2, and IL-18R are targets of miR-499 , and theIn summary, this is the first report demonstrating the relationship between miR-146a rs2910164 G>C and AS. We demonstrate that G alleles in the locus are associated with an increased risk for AS. This SNP may serve as a promising candidate marker for identification of AS. Furthermore, we hypothesize that this SNP may also be useful for identifying healthy people who are more prone to develop inflammatory disease. Further larger investigations are needed to verify this conclusion. If this is the case, an assessment of the rs2910164 during examinations of high-risk patients could lead to the early detection of AS. Whether the miR-499 rs3746444 gene is a non-significant factor in AS requires further validation. Our research sheds new light on the significant relationship between natural genetic variations in miRNA genes and human diseases. Studies with large cohorts and diverse ethnicities are warranted to validate our finding. In this field, further investigation on the expression level of miRNAs and different factors in the signal pathway will gradually reveal the underlying pathogenic mechanism of AS.